This invention relates generally to methods of modifying genes with specificity in recombination deficient cells by transiently enabling homologous recombination in the cells. Included in the invention are conditional replication shuttle vectors which bestow transient recombination capabilities to an otherwise recombination deficient cell. The independent origin based cloning vectors containing the modified genes and methods of using the independent origin based cloning vectors containing the modified genes are also included in the present invention. In particular, high throughput methodology is provided for generating the modified the independent origin based cloning vectors.
Functional analyses of genes in vivo frequently involve the introduction of modified genomic DNA into the germline to generate transgenic animals [Jaenisch et al., Science 240:1468 (1985); Brinster, Cell 41:343 (1985)]. The genomic DNA sequences containing introns and essential regulatory sequences have been shown to be expressed in vivo in cases where simple cDNA constructs cannot be expressed [Brinster et al., Proc.Natl.Acad.Sci. 85:836-840 (1988)]. Furthermore, the size of the genomic DNA that can be readily manipulated in vitro and introduced into the germline can be a critical determinant of the outcome of the functional analysis of a gene since elements that are important for high level, tissue specific and position-independent expression of the transgene may be located at a long distance from the gene itself [Dillon et al., Trends Genet. 9:134 (1993); Kennison, Trends Genet. 9:75 (1993); Wilson et al., Annu.Rev. Cell.Biol. 6:679 (1990)].
On the other hand, the use of such large genomic transgenes has several practical problems. For example, the size of the transgene is presently limited due to constraints on the sequence length that can be cloned and stably maintained in a conventional plasmid or a cosmid. Thus DNA sequences suspected of being nonessential are often omitted when designing the constructs to be transferred because of the size limitation. In addition, in vitro manipulations of large DNAs oftentimes lead to mechanical shear [Peterson et al., TIG 13:61-66].
Yeast artificial chromosomes (YACs) allow large genomic DNA to be modified and used for generating transgenic animals [Burke et al., Science 236:806; Peterson et al., Trends Genet. 13:61 (1997); Choi, et al., Nat. Genet., 4:117-223 (1993), Davies, et al., Biotechnology 11:911-914 (1993), Matsuura, et al., Hum. Mol. Genet., 5:451-459 (1996), Peterson et al., Proc. Natl. Acad. Sci., 93:6605-6609 (1996); and Schedl, et al., Cell, 86:71-82 (1996)]. Other vectors also have been developed for the cloning of large segments of mammalian DNA, including cosmids, and bacteriophage P1 [Sternberg et al., Proc. Natl. Acad. Sci. U.S.A., 87:103-107 (1990)]. YACs have certain advantages over these alternative large capacity cloning vectors [Burke et al., Science, 236:806-812 (1987)]. The maximum insert size is 35-30 kb for cosmids, and 100 kb for bacteriophage P1, both of which are much smaller than the maximal insert for a YAC. However, there are several critical limitations in the YAC system including difficulties in manipulating YAC DNA, chimerism and clonal instability [Green et al., Genomics, 11:658 (1991); Kouprina et al., Genomics 21:7 (1994); Larionov et al., Nature Genet. 6:84 (1994)]. As a result, generating transgenic mice with an intact YAC remains a challenging task [Burke et al., Science 236:806; Peterson et al., Trends Genet. 13:61 (1997)].
An alternative to YACs are E. coli based cloning systems based on the E. coli fertility factor that have been developed to construct large genomic DNA insert libraries. They are bacterial artificial chromosomes (BACs) and P-1 derived artificial chromosomes (PACs) [Mejia et al., Genome Res. 7:179-186 (1997); Shizuya et al., Proc. Natl. Acad. Sci. 89:8794-8797 (1992);Ioannou et al., Nat. Genet., 6:84-89 (1994); Hosoda et al., Nucleic Acids Res. 18:3863 (1990)]. BACs are based on the E. coli fertility plasmid (F factor); and PACs are based on the bacteriophage P1. The size of DNA fragments from eukaryotic genomes that can be stably cloned in Escherichia coli as plasmid molecules has been expanded by the advent of PACs and BACs. These vectors propagate at a very low copy number (1-2 per cell) enabling genomic inserts up to 300 kb in size to be stably maintained in recombination deficient hosts (most clones in human genomic libraries fall within the 100-200 kb size range). The host cell is required to be recombination deficient to ensure that non-specific and potentially deleterious recombination events are kept to a very minimum. As a result, libraries of PACs and BACs are relatively free of the high proportion of chimeric or rearranged clones typical in YAC libraries, [Monaco et al., Trends Biotechnol 12:280-286 (1994); Boyseu et al., Genome Research, 7:330-338 (1997)]. In addition, isolating and sequencing DNA from PACs or BACs involves simpler procedures than for YACs, and PACs and BACs have a higher cloning efficiency than YACs [Shizuya et al., Proc. Natl. Acad. Sci. 89:8794-8797 (1992);Ioannou et al., Nat. Genet., 6:84-89 (1994); Hosoda et al., Nucleic Acids Res. 18:3863 (1990)]. Such advantages have made BACs and PACs important tools for physical mapping in many genomes [Woo et al., Nucleic Acids Res., 22:4922 (1994); Kim et al., Proc.Natl.Acad.Sci. 93:6297-6301 (1996); Wang et al., Genomics 24:527 (1994); Wooster et al., Nature 378:789 (1995)]. Furthermore, the PACs and BACs are circular DNA molecules that are readily isolated from the host genomic background by classical alkaline lysis [Bimboim et al., Nucleic Acids Res. 7:1513-1523 (1979].
Functional characterization of a gene of interest contained by a PAC or BAC clone generally entails transferring the DNA into a eukaryotic cell for transient or long-term expression. A transfection reporter gene, e.g. a gene encoding lacZ, together with a selectable marker, e.g., neo, can be inserted into a BAC [Mejia et al., Genome Res. 7:179-186 (1997)]. Transfected cells can be then detected by staining for X-Gal to verify DNA uptake. Stably transformed cells are selected for by the antibiotic G418.
However, while PACs and BACs have cloning capacities up to 350 kb, performing homologous recombination to introduce mutations into a gene of interest has not been demonstrated [Peterson et al., TIG 13:61-66]. Indeed, although BACs or PACs have become an important source of large genomic DNA in genome research, there are still no methods available to modify the BACs or PACs. Furthermore, no germline transmission of intact BACs or PACs in transgenic mice have been reported. These, as well as other disadvantages of BACs and PACs greatly limit their potential use for functional studies. Therefore, there is a need for an improved cloning vector for germline transmission of selected genes in transgenic animals. More particularly there is a need for a cloning vector that has the capacity to contain greater than 100 kilobases of DNA, which can be readily manipulated and isolated, but still can be stably stored in libraries relatively free of rearranged clones. In addition, there is a need to provide methodology for generating such cloning vectors. There is also a need to apply such vectors to improve the results of the methods of gene transfer used in gene targeting, for creating animal models for diseases due to a dominant mutated allele, e.g., Huntington""s disease, and for overexpressing in vivo proteins encoded by genes having an unknown function in order to determine the biological role of such genes.
Gene targeting has been used in various systems, from yeast to mice, to make site specific mutations in the genome. Gene targeting is not only useful for studying function of proteins in vivo, but it is also useful for creating animal models for human diseases, and in gene therapy. The technique involves the homologous recombination between DNA introduced into a cell and the endogenous chromosomal DNA of the cell. However, in the vertebrate system, the rate of homologous recombination is very low, as compared to random integration. The only cell line that allows a relatively high homologous recombination rate and maintains the ability to populate the germline is the murine 129 embryonic stem cells (ES cells). Using this specialized cell, mice can be generated with a targeted mutation [Joyner, A. L., Gene Targeting: A Practical Approach. The Practical Approach Series (Rickwood, D., and Hames, B. D., Eds.), IRL Press, Oxford (1993)]. However, the rate of homologous recombination for some gene loci in ES cells is still extremely low ( less than 1%), the procedure is labor intensive, and the cost of generating targeted mutant mice is very expensive. Moreover, since there are no ES cells available for vertebrates other than mice, gene targeting in a germline is still not possible for other vertebrates.
A major limitation for gene transfer procedures in vertebrate cells such as gene targeting is the low targeting frequency. One critical factor affecting the targeting frequency is the total length of homology. Deng and Capecchi [MCB, 12:3365-3371 (1992)] have shown that gene targeting frequency is linearly-dependent on the logarithm of the total homology length over homology lengths of 2.8 kb to 14.6 kb. Since the curve did not plateau at the 14.6 kb homology, it is likely that incorporating greater homology lengths into the targeting vector will further increase the homologous recombination rate. Using a mathematical model developed by Fujitani et al, [Genetics, 140:797-809, (1995)], an estimate can be made that with a total homology of 100 kb isogenous DNA (i.e., DNA from the same strain of mice), the gene targeting rate in ES cells would be 10%. This is a dramatic improvement over the conventional 14.6 kb targeting vector, which only yields a corresponding rate of only 0.03%. Further support for the present strategy i.e., using a large DNA construct for gene targeting rate comes from an experiment with Mycobacterium tuberculosis, the causal agent of tuberculosis. Like vertebrate cells, gene targeting in TB has a very low rate, mainly due to the predominance of random integration over homologous recombination. It has been demonstrated that using a 40-50 kb linear targeting construct, a 6% targeting frequency could be obtained, whereas no targeting event was obtained at all with a smaller ( less than 10 kb) targeting construct [Balasubramanian et al., J. of Bacteriology 178:273-279 (1996)]. Therefore, there is a need to construct large gene transfer constructs to allow efficient gene transfer in many biological systems.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
The present invention provides a novel and efficient method of modifying independent origin based cloning vectors for in vitro and in vivo gene expression. In its broadest embodiment, the present invention provides a method of selectively performing homologous recombination on a particular nucleotide sequence contained in a recombination deficient host cell, i.e., a cell that cannot independently support homologous recombination. The method can employ a recombination cassette which contains a nucleic acid that selectively integrates into the particular nucleotide sequence when the recombination deficient host cell is induced to support homologous recombination. The method comprises introducing the recombination cassette into the recombination deficient host cell, and inducing the recombinantly deficient host cell to transiently support homologous recombination, thereby allowing the nucleic acid to integrate into the particular nucleotide sequence. In a preferred embodiment, unselected nucleotide sequence rearrangements and deletions, which are characteristic of host cells that support homologous recombination, are not evident with restriction endonuclease digestion map analysis with a restriction enzyme such as HindIII, EcoRI, XhoI, or AvrII. In a more preferred embodiment, unselected nucleotide sequence rearrangements and deletions are not evident with restriction endonuclease digestion map analysis with two or more restriction enzymes. In an important aspect of the present invention a high throughput methodology is provided for generating modified independent origin based cloning vectors e.g., BACs that comprise genomic DNA.
In a particular aspect of the present invention, the recombination deficient host cell cannot independently support homologous recombination because the host cell is RecA. In this aspect of the invention, inducing the host cell to transiently support homologous recombination comprises inducing the transient expression of a RecA-like protein in the host cell. In a preferred embodiment, inducing the transient expression of the RecA-like protein can be performed with a conditional replication shuttle vector. In a more preferred embodiment the conditional replication shuttle vector is a temperature sensitive shuttle vector (TSSV) that replicates at a permissive temperature, but does not replicate at a non-permissive temperature.
In one particular embodiment of this type, inducing the transient expression of the RecA-like protein comprises transforming the host cell with the TSSV at a permissive temperature, and growing the host cell at a non-permissive temperature. The TSSV encodes a RecA-like protein that is expressed in the host cell and supports the homologous recombination between a nucleic acid contained in a recombination cassette and the particular nucleotide sequence contained in the host cell. The TSSV encoding the RecA-like protein is diluted out when the host cell is grown at the non-permissive temperature. In one particular embodiment of this type the permissive temperature is 30xc2x0 C. and the non-permissive temperature is 43xc2x0 C.
In a more intricate version of the present invention, the particular nucleotide sequence which has been selected to undergo homologous recombination is contained in an independent origin based cloning vector (IOBCV) that is comprised by the host cell, and neither the independent origin based cloning vector alone, nor the independent origin based cloning vector in combination with the host cell, can independently support homologous recombination. In a particular embodiment of this type both the independent origin based cloning vector and the host cell are RecAxe2x88x92, and inducing the host cell to transiently support homologous recombination comprises inducing the transient expression of the RecA-like protein to support homologous recombination in the host cell. In one particular embodiment the independent origin based cloning vector is a Bacterial or Bacteriophage-Derived Artificial Chromosome (BBPAC) and the host cell is a host bacterium. In a preferred embodiment, inducing the transient expression of the RecA-like protein is performed with a conditional replication shuttle vector that encodes the RecA-like protein.
In one such embodiment the conditional replication shuttle vector contains an origin of DNA replication that requires the expression of a specific protein or proteins for replication that is (are) not normally present in host bacteria. In a particular embodiment of this type, the origin of DNA replication is the R6Kxcex3 DNA replication origin [oriR (R6Kxcex3)] and the specific protein that is expressed by the specific host cell is the pi replication protein which is encoded by the pir gene.
In another such embodiment the conditional replication shuttle vector is a temperature sensitive shuttle vector (TSSV) that replicates at a permissive temperature, but does not replicate at a non-permissive temperature. In one particular embodiment of this type the permissive temperature is 30xc2x0 C. and the non-permissive temperature is 43xc2x0 C. In another such embodiment the RecA-like protein is controlled by an inducible promoter and the transient expression of the RecA-like protein is achieved by the transient induction of the inducible promoter in the host cell. In another embodiment, the RecA-like protein is controlled by a constitutive promoter with the transient expression induced by the TSSV.
In a preferred embodiment the conditional replication shuttle vector contains a TSSV that also comprises a recombination cassette and a first gene which bestows resistance to a host cell that contains the TSSV against a first toxic agent. In addition, the first gene can be counter-selected against. The recombination cassette, the RecA-like protein gene, and the first gene are linked together on the TSSV such that when the nucleic acid integrates (i.e. resolved) into the particular nucleotide sequence, the RecA-like protein gene and the first gene remain linked together, and neither the RecA-like protein gene nor the first gene remain linked to the integrated nucleic acid. In a particular embodiment of this type, the independent origin based cloning vector is a BBPAC and the host cell is a bacterium. The BBPAC further contains a second gene that bestows resistance to the host cells against a second toxic agent. Introducing the recombination cassette into the host cells is performed by transforming the host cell with the TSSV. Inducing the transient expression of the RecA-like protein to support homologous recombination comprises: (i) incubating the host cells at a permissive temperature in the presence of the first toxic agent and the second toxic agent, wherein transformed host cells containing the TSSV and the BBPAC are selected for and wherein the RecA-like protein is expressed. A first homologous recombination event occurs between the recombination cassette and the particular nucleotide sequence forming a co-integrate between the TSSV and the BBPAC, wherein the TSSV is either free or part of a co-integrate; (ii) incubating the transformed host cells at a non-permissive temperature in the presence of the first toxic agent and the second toxic agent, wherein host cells containing a TSSV co-integrate are selected for, and wherein free TSSV cannot replicate; (iii) selecting a host cell containing a co-integrate between the TSSV and the BBPAC by Southern analysis; (iv) incubating the host cells containing a co-integrate between the TSSV and the BBPAC at a non-permissive temperature in the presence of the second toxic agent, wherein a second homologous recombination event occurs between the recombination cassette and the particular nucleotide sequence, therein integrating the nucleic acid into the particular nucleotide sequence and forming a resolved host cell, i.e., a host cell containing a resolved BBPAC; and (v) incubating the host cells containing the resolved BBPAC in the presence of the second toxic agent, and a counter-selecting agent, and wherein the counter-selecting agent is toxic to host cells containing the first gene, and wherein host cells containing the RecA-like protein gene are removed. Another embodiment further comprises selecting a host cell containing the resolved BBPAC by colony hybridization with a labeled probe that binds to a DNA homologue of the nucleic acid, an mRNA homologue of the nucleic acid, and/or a protein encoded by the nucleic acid. In a particular embodiment, the permissive temperature is 30xc2x0 C., the non-permissive temperature is 43xc2x0 C. In a preferred embodiment the incubating of host cells containing the resolved BBPAC in the presence of the second toxic agent and counter-selecting agent is performed at 37xc2x0 C. Preferred embodiments further comprise the generating of the recombination cassette by placing a first genomic fragment 5xe2x80x2 of the specific nucleic acid that is to selectively integrate into the particular nucleotide sequence, and placing a second genomic fragment 3xe2x80x2 of the specific nucleic acid. The first genomic fragment corresponds to a region of the particular nucleotide sequence that is 5xe2x80x2 to the region of the particular nucleotide sequence that corresponds to the second genomic fragment. Thus, both the first genomic fragment and the second genomic fragment contain portions of the particular nucleotide sequence. In one such embodiment, both the first genomic fragment and the second genomic fragment contain 250 or more basepairs of the particular nucleotide sequence. In a preferred embodiment, the first and second genomic fragments are about the same size. In another embodiment, both the first genomic fragment and the second genomic fragment contain 500 or more basepairs of the particular nucleotide sequence. In still another embodiment, both the first genomic fragment and the second genomic fragment contain 1000 or more basepairs of the particular nucleotide sequence. In one particular embodiment the recombination cassette is generated in a building vector and the recombination cassette is subsequently transferred to the TSSV.
In a particular embodiment the first gene confers tetracycline resistance and the counter-selecting agent is fusaric acid. In a preferred embodiment the RecA-like protein is recA. In the more preferred embodiment the TSSV is pSV1.RecA having the ATCC no. 97968.
In a related aspect of the present invention the RecA-like protein is controlled by an inducible promoter, and the transient expression of the RecA-like protein is achieved by the transient induction of the inducible promoter in the host cell. In one embodiment of this type, the independent origin based cloning vector is a BBPAC and the recombination deficient host cell is an E. coli bacterium. In a preferred embodiment the RecA-like protein is recA.
The present invention also provides a conditional replication shuttle vector that encodes a RecA-like protein. In one such embodiment the RecA-like protein is controlled by an inducible promoter. In a preferred embodiment the conditional replication shuttle vector is a temperature sensitive shuttle vector (TSSV). The RecA-like protein of the TSSV can be controlled by either a constitutive promoter or by an inducible promoter. In another embodiment the conditional replication shuttle vector contains an origin of DNA replication that requires the expression of a specific protein or proteins for replication that is (are) not normally present in host bacteria but is (are) in a specific host cell.
In one embodiment the conditional replication shuttle vector contains a gene that can be counter-selected against. In a specific embodiment of this type the conditional replication shuttle vector contains a gene that confers tetracycline resistance. In another embodiment the conditional replication shuttle vector contains a RecA-like protein that is recA. In still another embodiment the conditional replication shuttle vector contains both a gene that confers tetracycline resistance and a RecA-like protein that is recA. In a preferred embodiment the conditional replication shuttle vector is a TSSV. In a more preferred embodiment the TSSV is pSV1.RecA having the ATCC no. 97968.
The present invention further provides conditional replication shuttle vectors that comprise an R6Kxcex3 origin of replication and a nucleic acid encoding a recombination protein. In a preferred embodiment the recombination protein is recA. Preferably, the conditional replication shuttle vector is constructed so that it can modify a gene of interest in an IOBCV, preferably a BBPAC, and more preferably a BAC through homologous recombination. Such modifications include insertions, substitutions, and/or deletions. In a particular embodiment, the conditional replication shuttle vector further comprises a nucleic acid encoding one or more marker proteins or peptides that are to be inserted into the IOBCV so a particular gene product (encoded by the IOBCV) can be identified and/or monitored. In one such embodiment, the nucleic acid encodes the marker protein IRES-EGFP. In another embodiment, the nucleic acid encodes the marker FLAG peptide. In still another embodiment, the nucleic acid is taulacZ. In yet another embodiment, the nucleic acid is lacZ. As indicated above and exemplified below, multiple maker proteins/peptides can be encoded in the conditional replication shuttle vectors of the present invention and subsequently inserted into/or adjacent to the protein encodeded by the genc of interest.
In a preferred embodiment the conditional replication shuttle vector further comprises a gene that can be counter-selected against. In a preferred embodiment of this type, the gene that can be counter-selected against is SacB. In another embodiment, the gene that can be counter-selected against confers tetracycline resistance.
In one embodiment the conditional replication shuttle vector further comprises an A box region that either comprises or can be constructed to comprise a nucleic acid that can selectively integrate into a particular nucleotide sequence of a gene of interest contained by an IOBCV when the IOBCV and the conditional replication shuttle vector are placed in a host cell in which recombination events can occur. Preferably the A box region is bracketed by two restriction enzyme sites. Thus, the A box region and the restriction enzyme sites can be used to insert any selected nucleic acid into the conditional replication shuttle vector. In one particular embodiment, the two restriction enzyme sites are Asc1 and Sma1. Preferably the selected nucleic acid is between 300 and 500 basepairs, though substantially larger nucleic acids can be used when desired.
In a particular embodiment the conditional replication shuttle vector further comprises two frt sites. The two fit sites are positioned on opposite sides of the A box. Since the frt sites are used in the resolution step following the co-integration of the selected nucleic acid with the IOBCV, when it is desired to place one or more markers into the IOBCV, these markers are also positioned in the conditional replication shuttle vector in between the two frt sites.
In an alternative embodiment, the conditional replication shuttle vector further comprises two homologous nucleotide sequences, which are homologous to each other, but preferably are not homologous to the IOBCV that comprises the nucleotide sequence which forms the co-integrate with the selected nucleic acid of the conditional replication shuttle vector. Preferably the homologous nucleotide sequence is longer than the corresponding selected nucleic acid. In one such embodiment the homologous nucleotide sequence is greater than 500 basepairs. In another embodiment the homologous nucleotide sequence is greater than 1000 basepairs. In still another embodiment the homologous nucleotide sequence is greater than 5000 basepairs. As described above for the two frt sites, the two homologous nucleotide sequences are positioned on opposite sides of the A box. Again, since the two homologous nucleotide sequences are used in the resolution step following the co-integration of the selected nucleic acid with the IOBCV, when it is desired to place one or more markers into the IOBCV, these additional markers are also positioned on the conditional replication shuttle vector in between the two homologous nucleotide sequences. As exemplified below, the two homologous nucleotide sequences preferably encode one or marker proteins (and peptides). Thus, in a preferred embodiment the homologous nucleotide sequence encodes the enhanced green fluorescent protein (IRESEGFP).
As indicated above, the present invention provides methods of selectively performing homologous recombination with a particular nucleotide sequence of an independent origin based cloning vector (IOBCV) that is contained in a recombination deficient host cell. Such methods comprise introducing a conditional replication shuttle vector into a recombination deficient host cell and therein enabling homologous recombination in the host cell via the transient expression of a recombination protein in the host cell. The host cell comprises an IOBCV which contains the particular nucleotide sequence whereas the conditional replication shuttle vector encodes a recombination protein that is transiently expressed by the host cell. The conditional replication shuttle vector also contains a nucleic acid that selectively integrates into the particular nucleotide sequence when the recombination protein is expressed. Neither the IOBCV alone, nor the IOBCV in combination with the host cell can independently support homologous recombination.
The present invention further provides methods of selectively modifying a particular nucleotide sequence of an independent origin based cloning vector (IOBCV) that is contained in a recombination deficient host cell that are particularly conducive for high throughput procedures. These high throughput procedures are preferentially performed almost entirely in liquid rather than on plates thereby facilitating the modification of multiple BACs at one time, (e.g., performing separate modifications to different BACs at the same time).
One such embodiment comprises introducing a conditional replication shuttle vector into a recombination deficient host cell in which the host cell contains an IOBCV that comprises a gene of interest which contains a particular nucleotide sequence. The conditional replication shuttle vector encodes a recombination protein that is expressed by the host cell and permits homologous recombination to occur in the host cell since neither the IOBCV alone, nor the IOBCV in combination with the host cell can independently support homologous recombination. Preferably the recombination deficient host cell cannot independently support homologous recombination because the host cell is RecAxe2x88x92. In one embodiment the recombination protein is the rec E and rec T protein pair. In another embodiment the recombination protein is the Lambda beta protein. In yet another embodiment the recombination protein is the Arabidopsis thaliana DRT100 gene product. Preferably, the recombination protein is recA. The IOBCV is preferably a BBPAC and more preferably the BBPAC is a BAC.
The conditional replication shuttle vector contains a nucleic acid that selectively integrates into the particular nucleotide sequence when the recombination protein is expressed, thereby forming a co-integrate. The nucleic acid that selectively integrates into the particular nucleotide sequence and the nucleic acid encoding the recombination protein are positioned on the conditional replication shuttle vector such that upon resolution of the co-integrate, the nucleic acid encoding the recombination protein remains with the conditional replication shuttle vector. Thus, growing the host cell under conditions in which the conditional replication shuttle vector cannot replicate dilutes out the conditional replication shuttle vector encoding the recombination protein, and thereby prevents further (undesirable) recombination events in the recombination deficient cells to occur.
In a particular embodiment of this type, the conditional replication shuttle vector further comprises a nucleic acid that encodes a marker protein or peptide. The nucleic acid that selectively integrates into the particular nucleotide sequence and the nucleic acid encoding the marker protein or peptide are positioned on the conditional replication shuttle vector such that upon resolution of the co-integrate, the nucleic acid encoding the marker protein or peptide is inserted into or adjacent to the particular nucleotide sequence. In a particular embodiment, the conditional replication shuttle vector cannot replicate in the host cell because the conditional replication shuttle vector requires a particular protein for replication, and neither the host cell nor the IOBCV encode the particular protein. In a preferred embodiment of this type, the conditional replication shuttle vector cannot replicate in the host cell because the conditional replication shuttle vector comprises a R6Kxcex3 origin of replication and neither the host cell nor the IOBCV encode pir.
In a more preferred embodiment the conditional replication shuttle vector further comprises a first frt site that is positioned on one side of the nucleic acid that selectively integrates into the particular nucleotide sequence, and a second fit site that is positioned on the other side of the nucleic acid that selectively integrates into the particular nucleotide sequence. In this embodiment, the resolution of the co-integrate is performed by adding flip recombinase to the host cell. Flip recombinase is preferably added to the host cell by introducing a plasmid that encodes flip recombinase to the host cell. In a preferred embodiment, the plasmid contains a conditional origin of replication such as a temperature-sensitive origin of replication which allows the plasmid to be diluted out by growing the host cells at a temperature that disfavors the replication of the plasmid. The conditional replication shuttle vector can further comprise a nucleic acid encoding one or more marker proteins and/or peptides that are positioned in between the two frt sites and are also adjacent to the nucleic acid that selectively integrates into the particular nucleotide sequence, such that after the resolution, the marker protein(s) and/or peptide(s) are contained by the IOBCV.
Alternatively, the resolution step can be performed by a second homologous recombination step. In one such embodiment, the conditional replication shuttle vector further comprises two homologous nucleotide sequences that are homologous to each other but are not homologous to the IOBCV. The two homologous nucleotide sequences are positioned on the conditional replication shuttle vector to be on opposite sides of the nucleic acid that selectively integrates into the particular nucleotide sequence so that the resolution of the co-integrate is performed by a recombination event between the two homologous nucleotide sequences. As described above, since the two homologous nucleotide sequences are used in the resolution step following the co-integration of the selected nucleic acid with the IOBCV, when it is desired to place one or more markers into the IOBCV, these additional markers are also positioned on the conditional replication shuttle vector in between the two homologous nucleotide sequences. As exemplified below, the two homologous nucleotide sequences preferably encode one or marker proteins. Thus, in a preferred embodiment the homologous nucleotide sequence encodes the enhanced green fluorescent protein (e.g., IRESEGFP).
A more preferred embodiment further comprises adding a counterselection agent after the resolution of the co-integrate to remove host cells that comprise the conditional replication shuttle vector. In this case, the conditional replication shuttle vector is designed to further comprise a counterselection gene that is positioned on the conditional replication shuttle vector such that upon resolution of the co-integrate the counterselection gene remains with the conditional replication shuttle vector. In a preferred embodiment of this type the counterselection gene is SacB. In a more preferred embodiment of this type, the counterselection agent is sucrose.
The present invention also provides the independent origin based cloning vector that contains a particular nucleotide sequence that has undergone homologous recombination with a conditional replication shuttle vector in a RecA-host cell of the present invention. In a particular embodiment, the conditional replication shuttle vector encodes a RecA-like protein. The particular nucleotide sequence can be all or part of a given gene such as the gene that encodes the murine zinc finger gene, RU49 (also known as Zipro 1) as exemplified below. The nucleotide sequence can be constructed to further contain specific translational or transcription elements such as an IRES, and/or marker proteins such as the green fluorescent protein. In one preferred embodiment the independent origin based cloning vector has undergone homologous recombination with a temperature sensitive shuttle vector in a RecA-host cell, wherein the temperature sensitive shuttle vector encodes a RecA-like protein. In another embodiment the independent origin based cloning vector is a BBPAC, and more preferably a BAC. In a specific embodiment of this type the independent origin based cloning vector has undergone homologous recombination with a temperature sensitive shuttle vector that is pSV1.RecA having the ATCC no. 97968.
The present invention also provides methods of using the modified independent origin based cloning vectors of the present invention to make transgenic animals including making animal models for diseases due to a dominant mutated allele, e.g., Huntington""s disease; perform gene targeting; perform gene therapy; or for overexpressing in vivo proteins encoded by genes having an unknown function in order to determine the biological role of such genes, as exemplified below. The independent origin based cloning vectors or linearized nucleic acid inserts derived from the IOBCVs, for example, can be introduced into a eukaryotic cell or animal. In one such embodiment the transgenic animal made has a particular phenotype as a result of introducing (e.g., by pronuclear injecting) a BBPAC into the transgenic animal (or a fertilized zygote) which corresponds to a symptom of a particular disease. In this case, the BBPAC had been modified to contain a dominant allele known to be associated with and/or due to the particular disease.
In a related embodiment a BBPAC is identified that contains the wildtype copy of a gene that has been associated with one or more symptoms of a particular disease when the nucleotide sequence of the gene has a particular modification. In one such embodiment the BBPAC containing the wildtype gene is modified through homologous recombination by a method of the present invention, e.g. with a conditional replication shuttle vector, so that it contains the nucleotide sequence that has been associated with one or more symptoms of the particular disease. The modified BBPAC is then placed into a transgenic animal or a eukaryotic cell (e.g., a fertilized zygote) which results in a transgenic animal that has a phenotype that can be correlated with one or more symptoms of the particular disease. The transgenic animal can then be used as an animal model for the particular disease.
In one such embodiment the eukaryotic cell is a fertilized zygote. In another embodiment the eukaryotic cell is a mouse ES cell. The gene targeting, for example, can be performed to modify a particular gene, or to totally disrupt the gene to form a knockout animal. Similarly, IOBCVs made by the methods disclosed herein can be added in multiple copies to a fertilized mammalian zygote for example, in order to achieve overexpression of a particular protein. In addition, an IOBCV made by the methods disclosed herein can be used to make an animal model for a particular disease in which the expression of a mutated allele (carried by the IOBCV) leads to the desired phenotype for the animal model.
Thus in one aspect of the present invention, the independent origin based cloning vector contains a nucleic acid that has undergone homologous recombination with a conditional replication shuttle vector in a RecAxe2x88x92 whole cell, in which the conditional replication shuttle vector includes a RecA like protein. In a preferred embodiment the independent origin based cloning vector is a BBPAC. In a more preferred embodiment, the BBPAC has undergone homologous recombination with a TSSV. In the most preferred embodiment, the BBPAC has undergone homologous recombination with the TSSV that ispSVl.RecA having the ATCC no. 97968.
One particular embodiment is a method of using the BBPAC to introduce the nucleic acid into an animal to make a transgenic animal comprising pronuclear injecting of the BBPAC (or a linearized nucleic acid insert derived from the BBPAC) into a fertilized zygote. In one embodiment the animal is a mammal. In a more preferred embodiment the mammal is a mouse. In a specific embodiment of this type the independent origin based cloning vector is a BBPAC and the fertilized zygote is a C57BL/6 mouse zygote. In a preferred embodiment of this type two picoliters (pl) of less than one xcexcg/ml BBPAC DNA is injected. In a more preferred embodiment 2 pl of 0.6 xcexcg/ml of DNA is injected.
The present invention also includes a method of using the BBPAC of the invention to perform gene targeting in a vertebrate cells comprising introducing the BBPAC into the vertebrate cell wherein the nucleic acid that has undergone homologous recombination with the conditional replication shuttle vector, undergoes homologous recombination with the endogenous chromosomal DNA of the vertebrate cell. In preferred embodiments of this type the vertebrate cell is a mammalian cell. In a more preferred embodiment of this type the mammalian cell is a human cell. In a related embodiment the vertebrate cell is a fertilized zygote and the nucleic acid contains a disrupted gene. In a preferred embodiment the conditional replication shuttle vector is a TSSV. In a more preferred embodiment the TSSV is pSV1.RecA having the ATCC no. 97968.
The IOBCVs (including BBPACs and BACs) that have been modified by the methods of the present invention are also part of the present invention. The present invention further provides methods of producing non-human transgenic animals using these IOBCVs. One such method comprises introducing the IOBCV into a eukaryotic cell and placing the eukaryotic cell into a recipient animal, whereby the eukaryotic cell develops into the non-human transgenic animal. In one such embodiment, the eukaryotic cell is a fertilized animal zygote. In another embodiment the eukaryotic cell is an embryonic stem cell. In another embodiment, the eukaryotic cell is an ES-like cell. In addition, all of the non-human transgenic animals generated by such methodology are also part of the present invention.
The present invention also contains kits for performing homologous recombination on selected nucleotide sequences contained on an independent origin based cloning vector, such as a BBPAC. Any of the shuttle vectors of the present invention can be included in the kits. In one particular embodiment, the kit comprises a conditional replication shuttle vector and a building vector. In a preferred embodiment of this type, the kit further contains a restriction map for the shuttle vector and/or a restriction map for one or more of the building vectors. In a more preferred embodiment, the kit further includes a protocol for using the contents of the kit to perform homologous recombination.
A particular embodiment of the kit contains a TSSV, such as pSV1.RecA and a building vector. In one such embodiment the building vector is pBV.IRES.LacZ.PA. In another such embodiment the building vector is pBV.EGFP1. In yet another such embodiment the building vector is pBV.IRES.EGFP1. In still another such embodiment the building vector is pBV.pGK.Neo.PA.
In a preferred embodiment two or more building vectors are included in the kit. In a more preferred embodiment all four of the above-listed building vectors are included in the kit. Restriction maps for one or more of the building vectors or the TSSV may also be included in the kits. In addition, the kits may also include a protocol for using the contents of the kit to perform homologous recombination. In one specific embodiment, a kit contains pSV1.RecA and one or more of the above-listed vectors also contains fusaric acid and/or chloro-tetracycline.
Accordingly, it is a principal object of the present invention to provide a method for readily and specifically modifying an independent origin based cloning vector in a recombination deficient host cell.
It is a further object of the present invention to provide a method of transiently expressing a RecA-like protein in a RecA-host cell to allow the specific modification of a gene of interest contained by an independent origin based cloning vector.
It is a further object of the present invention to provide a method of generating deletions, substitutions, and/or point mutations in a specific gene contained by the independent origin based cloning vector in a RecAxe2x88x92 cell.
It is a further object of the present invention to provide a conditional replication shuttle vector which encodes a RecA-like protein, and which further contains a specific nucleic acid in a recombination cassette that selectively undergoes homologous recombination with an independent origin based cloning vector when both vectors are present in a recombination deficient host cell.
It is a further object of the present invention to provide a temperature dependent shuttle vector which encodes a RecA-like protein.
It is a further object of the present invention to provide a temperature dependent shuttle vector which encodes a RecA-like protein, which further contains a specific nucleic acid in a recombination cassette that can selectively undergo homologous recombination with a gene of interest contained by an independent origin based cloning vector, when both vectors are placed in a recombination deficient host cell.
It is a further object of the present invention to provide a temperature sensitive shuttle vector that is pSV1.RecA having the ATCC no. 97968.
It is a further object of the present invention to provide a modified independent origin based cloning vector that can be used for the pronuclear injection of a nucleic acid contained by IOBCV into an animal zygote.
It is a further object of the present invention to provide a modified independent origin based cloning vector that can be transfected into an embryonic stem cell.
It is a further object of the present invention to provide a method of introducing a linearized nucleic acid insert from a modified independent origin based cloning vector into a fertilized zygote of an animal.
It is a further object of the present invention to provide a method of introducing a modified independent origin based cloning vector into an embryonic stem cell.
It is a further object of the present invention to provide a method of purifying a large linearized BBPAC.
It is still a further object of the present invention to provide a method for readily and specifically modifying an independent origin based cloning vector in a recombination deficient host cell under conditions that allow multiple modifications of IOBCVs at the same time.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.